Antimicrobial factors form one arm of the innate immune system which protects mucosal surfaces from bacterial infection. These factors can rapidly kill bacteria deposited on mucosal surfaces and prevent acute invasive infections. In many chronic infections, however, bacteria live in biofilms; i.e., distinct, matrix-encased communities specialized for surface persistence. The transition from a free-living, independent existence to a biofilm lifestyle can be devastating because biofilms notoriously resist killing by host defense mechanisms and antibiotics.
Biofilms are defined as an association of microorganisms, e.g., single or multiple species, that grow attached to a surface and produce a slime layer that provides a protective environment (Costerton, J. W. (1995) J Ind. Microbiol. 15(3):137-40, Costerton, J. W. et al. (1995) Annu Rev Microbiol. 49:711-45). Typically, biofilms produce large amounts of extracellular polysaccharides, responsible for the slimy appearance, and are characterized by an increased resistance to antibiotics (1000- to 1500-fold less susceptible). Several mechanisms are proposed to explain this biofilm resistance to antimicrobial agents (Costerton, J. W. et al. (1999) Science. 284(5418):1318-22).
One theory is that the extracellular matrix in which the bacterial cells are embedded provides a barrier toward penetration by the biocides. A further possibility is that a majority of the cells in a biofilm are in a slow-growing, nutrient-starved state, and therefore not as susceptible to the effects of anti-microbial agents. A third mechanism of resistance could be that the cells in a biofilm adopt a distinct and protected biofilm phenotype, e.g., by elevated expression of drug-efflux pumps.
In most natural settings, bacteria grow predominantly in biofilms. Biofilms of P. aeruginosa have been isolated from medical implants, such as indwelling urethral, venous or peritoneal catheters (Stickler, D. J. et al. (1998) Appl Environ Microbiol. 64(9):3486-90). Chronic P. aeruginosa infections in cystic fibrosis lungs are considered to be biofilms (Costerton, J. W. et al. (1999) Science. 284 (5418):1318-22). P. aeruginosa is also of great industrial concern (Bitton, G. (1994) Wastewater Microbiology. Wiley-Liss, New York, N.Y.; Steelhammer, J. C. et al. (1995) Indust. Water Treatm. :49-55). The organism grows in an aggregated state, the biofilm, which causes problems in many water-processing plants. Of particular public health concern are food processing and water purification plants. Problems include corroded pipes, loss of efficiency in heat exchangers and cooling towers, plugged water injection jets leading to increased hydraulic pressure, and biological contamination of drinking water distribution systems (Bitton, G. (1994) Wastewater Microbiology. Wiley-Liss, New York, N.Y., 9). The elimination of biofilms in industrial equipment has so far been the province of biocides. Biocides, in contrast to antibiotics, are antimicrobials that do not possess high specificity for bacteria, so they are often toxic to humans as well. Biocide sales in the US run at about $1 billion per year (Peaff, G. (1994) Chem. Eng. News: 15-23).
A particularly ironic connection between industrial water contamination and public health issues is an outbreak of P. aeruginosa peritonitis that was traced back to contaminated poloxamer-iodine solution, a disinfectant used to treat the peritoneal catheters. P. aeruginosa is commonly found to contaminate distribution pipes and water filters used in plants that manufacture iodine solutions. Once the organism has matured into a biofilm, it becomes protected against the biocidal activity of the iodophor solution. Hence, a common soil organism that is harmless to the healthy population, but causes mechanical problems in industrial settings, ultimately contaminated antibacterial solutions that were used to treat the very people most susceptible to infection.
P. aeruginosa is a soil and water bacterium that can infect animal hosts. Normally, the host defense system is adequate to prevent infection. However, in immunocompromised individuals (such as burn patients, patients with cystic fibrosis, or patients undergoing immunosuppressive therapy), P. aeruginosa is an opportunistic pathogen, and infection with P. aeruginosa can be fatal (Govan, J. R. et al. (1996) Microbiol Rev. 60(3):539-74; Van Delden, C. et al. (1998) Emerg Infect Dis. 4(4): 551-60).
For example, Cystic fibrosis (CF), the most common inherited lethal disorder in Caucasian populations (˜1 out of 2,500 life births), is characterized by bacterial colonization and chronic infections of the lungs. The most prominent bacterium in these infections is P. aeruginosa. By their mid-twenties, over 80% of people with CF have P. aeruginosa in their lungs (Govan, J. R. et al. (1996) Microbiol Rev. 60(3):539-74). Although these infections can be controlled for many years by antibiotics, ultimately the P. aeruginosa bacteria form a biofilm that is resistant to antibiotic treatment. At this point the prognosis is poor. The median survival age for people with CF is the late 20s, with P. aeruginosa being the leading cause of death (Govan, J. R. et al. (1996) Microbiol Rev. 60(3):539-74). According to the Cystic Fibrosis Foundation, treatment of CF cost more than $900 million in 1995.
In addition, about two million Americans suffer serious burns each year, and 10,000-12,000 die from their injuries. The leading cause of death is infection (Lee, J. J. et al. (1990) J Burn Care Rehabil. 11(6):575-80). P. aeruginosa bacteremia occurs in 10% of seriously burned patients, with a mortality rate of 80% (Mayhall, C. G. (1993) p. 614-664, Prevention and control of nosocomial infections. Williams & Wilkins, Baltimore; McManus, A. T et al. (1985) Eur J Clin Microbiol. 4(2):219-23).
Such infections are often acquired in hospitals (“nosocomial infections”) when susceptible patients come into contact with other patients, hospital staff, or equipment. In 1995 there were approximately 2 million incidents of nosocomial infections in the U.S., resulting in 88,000 deaths and an estimated cost of $4.5 billion (Weinstein, R. A. (1998) Emerg Infect Dis. 4(3):416-20). Of the AIDS patients mentioned above who died of P. aeruginosa bacteremia, more than half acquired these infections in hospitals (Meynard, J. L. et al. (1999) J Infect. 38(3):176-81).
Nosocomial infections are especially common in patients of intensive care units as these people often have weakened immune systems and are frequently on ventilators and/or catheters. Catheter-associated urinary tract infections are the most common nosocomial infection (Richards, M. J. et al. (1999) Crit Care Med. 27(5):887-92) (31% of the total), and P. aeruginosa is highly associated with biofilm growth and catheter obstruction. While the catheter is in place, these infections are difficult to eliminate (Stickler, D. J. et al. (1998) Appl Environ Microbiol. 64(9):3486-90). The second most frequent nosocomial infection is pneumonia, with P. aeruginosa the cause of infection in 21% of the reported cases (Richards, M. J. et al. (1999) Crit Care Med. 27(5):887-92). The annual costs for diagnosing and treating nosocomial pneumonia has been estimated at greater than $2 billion (Craven, D. E. et al. (1991) Am J Med. 91(3B):44S-53S).
Treatment of these so-called nosocomial infections is complicated by the fact that bacteria encountered in hospital settings are often resistant to many antibiotics. In June 1998, the National Nosocomial Infections Surveillance (NNIS) System reported increases in resistance of P. aeruginosa isolates from intensive care units of 89% for quinolone resistance and 32% for imipenem resistance compared to the years 1993-1997 (NNIS. www.cdc.gov/ncidod/hip/ NNIS/AR_Surv 1198.htm). In fact, some strains of P. aeruginosa are resistant to over 100 antibiotics (Levy, S. (March 1998) Scientific American.). There is a critical need to overcome the emergence of bacterial strains that are resistant to conventional antibiotics (Travis, J. (1994) Science. 264:360-362).
Methods of inhibiting biofilm formation in medical and industrial settings have previously been developed using metal chelators. These methods have disclosed the use of small molecule chelators, i.e., EDTA, EGTA, deferoxamine, detheylenetriamine penta acetic acid and etidronate for the inhibition of biofilm. For example, U.S. Pat. No. 6,267,979 discloses the use of metal chelators in combination with antifungal or antibiotic compositions for the prevention of biofouling in water treatment, pulp and paper manufacturing, and oil field water flooding; U.S. Pat. No. 6,086,921 discloses the use of thiol containing compounds in combination with heavy metals as biocides; and U.S. Pat. No. 5,688,516 discloses the use of non-glycopeptide antimicrobial agents in combination with divalent metal chelating agents for use in the treatment and preparation of medical indwelling devices.
There still exists a need in the medical, environmental and industrial community for the control of biofilm formation. The control of biofilms needs to begin at the level of biofilm formation because, once formed, biofilms are exquisitely resistant to all common bactericidal methods. The present invention provides methods and compositions for inhibiting biofilm formation by chelating metal ions and provides methodology for the treatment of subjects with a bacterial infection prior to biofilm development.